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A) Sequence alignment between Repo-Man wt and the IRCE mutant generated. B) GFP pull-down on HeLa cells transfected with either GFP or GFP:RepoMan wt or GFP:RepoMan IRCE. The samples were run on SDS PAGE, blotted and probed with either GFP antibody (left panel) or anti SUMPO2/3 antibody (right panel). UB=unbound fraction B=bound fraction. C) HeLa cells were transfected with either GFP or GFP:RepoManwt. The GFP constructs were pulled-down with GFP-binder beads and incubated with recombinant purified lamin A. Western blot of the unbound (UB) and bound (B) fractions probed with anti GFP (Bottom panel), lamin A (middle panel) and RepoMan (top panel) antibodies. D) Quantification of the mitotic progression (1���3 h from release) of the HCT116 RepoMan:FKBP:GFP (green) / pMito-mCherry-FRB (red) cell line with and without rapamycin from the experiment designed in Figure 3 D. between 75���100 cells were analysed per each condition. The datasets with and without rapamycin at each time point were statistically analysed with a Chi Square test. Ns= not significant.

A) Representative images of HeLa cells stained for lamin A S22ph (red) and DAPI. A metaphase (top panel), an ana/telophase (middle panel) and a cytokinesis (bottom panel) are shown. Scale bar 10 ��m. B) Representative images of the experiment quantified in Figure 3 E. HeLa cells were treated with RepoMan siRNA oligos, transfected with either the oligo-resistant versions of GFP:RepoManwt or GFP:RepoManIRCE mutant (green) for 36h then fixed and stained for anti lamin A S22ph (red). C) Representative of the experiment quantified in Figure 3 G. PLA conducted in HeLa cells transfected with either GFP:RepoManwt or GFP:RepoManIRCE mutant. The primary antibodies were anti GFP and anti laminAS22.

A) GFP pull-down on HeLa cells transfected with either GFP:RepoMan wt or GFP:RepoMan IRCE or GFP:Repo-Man S893D. The samples were run on SDS PAGE, blotted and probed with either GFP antibody (bottom panels) of anti SUMO2/3 antibody (top panels). B=bound fraction. B) Representative images of HeLa cells transfected with either GFP:RepoMan wt (top panel), GFP:RepoMan S893D (middle panel) or GFP lamin A (bottom panel) (green). In yellow is the line scan in ImageJ used to quantify the distribution of the green signal. The profiles are represented in the graph on the right of each panel. The red arrows represent the increase in signal on the chromatin for Repo-Man wt and the decrease in signal on the chromatin for lamin A. However, the GFP:RepoMan S893D mutant is not showing exclusion form the chromatin but rather lack of accumulation.

Repo-Man is a protein phosphatase 1 (PP1) targeting subunit that regulates mitotic progression and chromatin remodelling. After mitosis, Repo-Man/PP1 remains associated with chromatin but its function in interphase is not known. Here we show that Repo-Man, via Nup153, is enriched on condensed chromatin at the nuclear periphery and at the edge of the nucleopore basket. Repo-Man/PP1 regulates the formation of heterochromatin, dephosphorylates H3S28 and it is necessary and sufficient for heterochromatin protein 1 binding and H3K27me3 recruitment. Using a novel proteogenomic approach, we show that Repo-Man is enriched at subtelomeric regions together with H2AZ and H3.3 and that depletion of Repo-Man alters the peripheral localization of a subset of these regions and alleviates repression of some polycomb telomeric genes. This study shows a role for a mitotic phosphatase in the regulation of the epigenetic landscape and gene expression in interphase., Repo-Man is a chromosome-binding subunit of protein phosphatase 1 to regulate mitosis. Here, de Castro and colleagues show that Repo-Man also regulates heterochromatin during interphase, and regulates gene repression and chromatin organization.

Frizzled receptors mediate Wnt ligand signalling, which is crucially involved in regulating tissue development and differentiation, and is often deregulated in cancer. In this study, we found that the gene encoding the Wnt receptor frizzled 6 (FZD6) is frequently amplified in breast cancer, with an increased incidence in the triple‐negative breast cancer (TNBC) subtype. Ablation of FZD6 expression in mammary cancer cell lines: (1) inhibited motility and invasion; (2) induced a more symmetrical shape of organoid three‐dimensional cultures; and (3) inhibited bone and liver metastasis in vivo. Mechanistically, FZD6 signalling is required for the assembly of the fibronectin matrix, interfering with the organization of the actin cytoskeleton. Ectopic delivery of fibronectin in FZD6‐depleted, triple‐negative MDA‐MB‐231 cells rearranged the actin cytoskeleton and restored epidermal growth factor‐mediated invasion. In patients with localized, lymph node‐negative (early) breast cancer, positivity of tumour cells for FZD6 protein identified patients with reduced distant relapse‐free survival. Multivariate analysis indicated an independent prognostic significance of FZD6 expression in TNBC tumours, predicting distant, but not local, relapse. We conclude that the FZD6–fibronectin actin axis identified in our study could be exploited for drug development in highly metastatic forms of breast cancer, such as TNBC. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.